Branched-chain amino acid aminotransferase (BCAT) is the enzyme responsible for catalyzing the first step in the metabolism of branched-chain amino acids (BCAAs) such as leucine, isoleucine, and valine. The step involves the reversible transamination of BCAAs to corresponding branched-chain α-keto acids (BCKAs). BCAT exists in two forms: a cytosolic form (BCAT1) and a mitochondrial form (BCAT2). The two isozymes exhibit distinct and non-overlapping distribution. While BCAT2 is considered ubiquitous, BCAT1 has limited expression and is thought to be found only in embryonic tissues, in adult brain, ovary, and placenta and in c-myc-induced brain tumors and T-cell lymphomas but not in c-myc-induced mammary tumors or B-cell lymphomas (Benvenisty N. et al., An embryonically expressed gene is a target for c-Myc regulation via the c-Myc-binding sequence. Genes Dev. (1992) 6:2513-2523).
Expression of the gene which encodes BCAT1 (Bcat1/Eca39) has been associated with proliferation in yeast and increased metastatic potential in human cancers. Schuldiner et al. showed that disruption of the Eca39 homologue in Saccharomyces cerevisiae resulted in increased proliferation (Schuldiner O. et al., ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast. Proc. Natl. Acad. Sci. USA (1996) 93:7743-7748). Eden and Benvenisty suggested that BCAT1 inhibits proliferation through production of α-ketoisocaproate, a leucine metabolite, which was shown to induce apoptotic death (Eden A. and Benvenisty N., Involvement of branched-chain amino acid aminotransferase (Bcat1/Eca39) in apoptosis. FEBS Let. (1999) 457:255-261).
Despite the recent advances in designing more effective cancer treatments, cancer remains the second leading cause of death in United States and a major health issue. Metastatic disease, as opposed to primary tumor growth, is usually the cause of cancer mortality. Despite this well-known fact, the majority of new anticancer drugs entering the marketplace focus on reducing tumor burden. It is apparent that what is needed is new drugs that prevent cancer invasion and inhibit cancer metastasis.
Metastatic colonization is governed by the reciprocal interaction of tumor cells with their host microenvironment, which consists of extracellular matrix (ECM) and normal cells such as fibroblasts, endothelial cells, or infiltrating inflammatory cells. The net outcome of this interaction is the production of growth factors, chemokines, and proteases such as vascular endothelial growth factor (VEGF) and extracellular matrix metalloproteinases (MMPs), which collectively facilitate tissue remodeling to allow for metastatic growth. Production of MMPs by tumor and host cells is greatly influenced by CD147, a ubiquitously expressed cell surface glycoprotein. CD147, also known as basigin or extracellular matrix metalloproteinase inducer (EMMPRIN), is grossly upregulated in cancer, where is thought to promote invasion, metastasis, growth, and survival of malignant cells through a multitude of functions. Specifically, cell surface expressed CD147: a) mediates both heterotypic and homotypic cell-cell interactions among tumor cells, fibroblasts, and endothelial cells resulting in synthesis and secretion of MMPs and VEGF, which, in turn, promote tumor angiogenesis, and b) supports anchorage-independent growth of cancer cells and renders tumor cells resistant to anoikis by downregulating Bim, a pro-apoptotic protein. CD147 is also known to: a) interact with cytoskeletal proteins and participate in the cytoskeleton rearrangement and cell motility, and b) associate with monocarboxylate transporters (MCTs) and facilitate their trafficking to the plasma membrane.
Dysregulation of inflammation, the physiological response to injury, is the hallmark of many autoimmune diseases including but not limited to systemic lupus erythematosus, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, and inflammatory bowel disease. Autoimmune inflammatory diseases are often characterized by elevation of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and interferon-alpha (IFN-α). The same cytokines may also play a role in the pathogenesis of these diseases (Postal M, Appenzeller S. The role of tumor necrosis factor-alpha (TNF-α) in the pathogenesis of systemic lupus erythematosus. Cytokine (2011) 56:537-543).
RA is a chronic, systemic autoimmune inflammatory disease that affects 0.5-1% of the world's adult population, most commonly women between the ages of 30 to 55 years old. It involves the significant alteration of the synovium and leads to musculoskeletal disability and an increased risk of mortality. Synovium is the thin cell layer which envelopes the cavity of synovial joints and produces synovial fluid for joint lubrication. It is composed of two morphologically distinct cell types: the macrophage-like synoviocytes (MLS) and the fibroblast-like synoviocytes (FLS). During RA, CD4+T cells along with macrophages and to a lesser degree B cells, plasma cells, dendritic and mast cells accumulate and invade through the synovial membrane, where they secrete pro-inflammatory signals and assist in the activation of the synovial cells. Secretion of pro-inflammatory cytokines from MLS leads to activation of FLS. Activated FLS aggressively degrade extracellular matrix alone or in co-operation with MLS. Hyperplasia of synoviocytes results in thickening of the synovial lining and its transformation into panus, a tissue mass that invades into articular cartilage and subchondral bone. Invading FLS degrade cartilage through secretion of MMPs and cathepsins. In addition, activated FLS and T cells release receptor activator of NF-κB ligand (RANKL), the protein responsible for the terminal differentiation of precursor myeloid cells into osteoclasts. In advanced RA, increased osteoclastogenesis leads to bone erosion (Pettit A R et al., RANKL protein is expressed at the pannus-bone interface at sites of articular bone erosion in rheumatoid arthritis. Rheumatology (Oxford) (2006) 45:1068-1076).
A number of options for the treatment of RA exist including non-steroidal anti-inflammatory drugs (NSAIDS), non-biologic disease-modifying anti-rheumatic drugs (DMARDs), and biologic DMARDs (Quan L D et al., Expert Opin Ther Pat (2008) 18(7):723-738). However, each of these options has drawbacks. NSAIDs provide immediate relief from pain and stiffness but are unable to halt the clinical progression of the disease. Biologic DMARDs such as those aiming at inhibiting TNFα are injectable and expensive drugs that exhibit long t1/2 and, thus, not easily eliminated in the case of toxicity. Toxic effects associated with the use of DMARDs include life-threatening infections and development of leukemia. It is apparent that what is desired is the development of new anti-rheumatic drugs that can be given orally, are inexpensive, and exhibit short t1/2. Since the majority of DMARDs treat RA by reducing inflammation without protecting against bone loss, it is further apparent that what is needed is anti-arthritic agents that can inhibit inflammation while inhibiting bone damage.
Bone homeostasis is maintained by two distinct cell populations: the bone-resorbing osteoclasts and the bone-forming osteoblasts. Normal bone remodeling is influenced by several factors including the RANKL/RANK pathway. RANKL, released by osteoblasts, binds to its cognate receptor RANK present on pre-osteoclastic cells such as monocytes/macrophages and induces their differentiation into mature osteoclasts (Silva I, Branco J C, Rank/Rankl/opg: literature review. Acta Rheumatol. Port. (2011) 36(3):209-218). RANKL overproduction leads to an imbalance between the amount of bone removed by osteoclasts and bone formed by osteoblasts and results in decreased skeletal integrity and risk of fracture. Bone diseases mediated by RANKL include but not limited to osteoarthritis, osteoporosis, and periodontal disease, giant cell tumor of the bone, and bone loss associated with chronic obstructive pulmonary disease. These pathological conditions are often associated with elevated levels of circulating RANKL.